Abstract

1361

Protein degradation is mediated predominantly through the ubiquitin proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The first generation proteasome inhibitor, bortezomib, is now an approved drug for the treatment of multiple myeloma. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening due to interference from test compounds. To address these issues, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. We first developed this method for monitoring caspase activities and have now developed homogeneous, bioluminescent assays for all three proteasome activities. We use Suc-LLVY-aminoluciferin, Z-LRR-aminoluciferin, and Z-nLPnLD-aminoluciferin to monitor the chymotrypsin-like, trypsin-like, and caspase-like activities of the proteasome, respectively. The luminogenic assays showed increased dynamic range and improved detection sensitivity compared to the analogous fluorogenic assays. In addition, the luminescent assays rapidly attained maximum signal and held stable light emission, allowing for assay flexibility. We have applied this technology to a cell-based assay using the Suc-LLVY-aminoluciferin substrate in combination with a selective membrane permeabilization step. The cell-based assay does not require lysate preparation and enables measurement of proteasome activity in a more physiologically relevant environment. The assay has been tested on numerous cell lines and has been shown to monitor proteasome activity that is inhibited by lactacystin and epoximicin. The proteasome assays are designed in a simple “add and read” format and have been tested in 96 and 384-well formats. The homogeneous, luminogenic protease assay format was at least 10-fold more sensitive than fluorogenic assays using comparable substrates for several proteases. The bioluminescent, coupled-enzyme format enables sensitive and rapid protease assays ideal for high-throughput screening.